At the cellular level, obesity is characterized by excess triacylglycerol (TAG) accumulation in adipose and non-adipose tissues. Glycerol-3-phosphate acyltransferase 1 (GPAT1) is critical for regulating hepatic TAG synthesis and mediating the development of insulin resistance. The mounting evidence from the discordant GPAT1 mRNA expression, GPAT1 protein level, and GPAT1 activity in hepatocytes strongly suggests a post- translational regulation mechanism. Furthermore, GPAT1 is phosphorylated by protein kinase CK2 and AMPK in vitro concomitant with an increase and decrease in GPAT1 activity, respectively. We hypothesize that GPAT1 is regulated in the liver in a phosphorylation-specific manner, and that these phosphorylated sites are physiologically relevant to GPATI's role in the development of insulin resistance. Current methodologies to study the phosphorylation-regulation of GPAT1 are incapable of providing site-specific evidence of GPAT1 phosphorylation and the corresponding function relevance. Due to its critical role in TAG synthesis, the function of modified GPAT1 is requisite for a fundamental understanding of the TAG synthetic pathway in the liver and the development of insulin resistance. Mass spectrometry measures an intrinsic property of molecule (mass) with extraordinary sensitivity, making it an unparalleled technology for site- specific phosphorylation mapping. Therefore, we propose an interdisciplinary approach to develop a robust, analytical method using mass spectrometry for the site-specific phosphorylation mapping of GPAT1 to assess the nutrient- and disease-mediated changes in the regulation of TAG synthesis. The specific aims of the proposal are 1) site-specific mapping of phosphorylated GPAT1 residues by mass spectrometry, 2) determine the functional relevance of AMPK-mediated changes in GPAT1 phosphorylation by measuring GPAT1 activity and phosphorylation status in hepatocytes when AMPK is activated or inhibited, and 3) identify changes in phosphorylation of endogenous GPAT1 mediated by feeding status (fasting, fasting/re- feeding) and disease states (obesity and type 2 diabetes mellitus). We expect endogenous GPAT1 phosphorylation at AMPK sites will decrease in the obese state and with fasting/high-carbohydrate refeeding of wild-type mice. Alternatively, endogenous GPAT1 phosphorylation at AMPK sites will increase with fasting in wild-type mice and upon metformin treatment in type 2 diabetic mice (when AMPK is activated). PUBLIC HEALTH RELEVANCE: Dyslipidemia is linked to a number of health conditions, including diabetes and obesity. The research proposed aims to further characterize the regulation of triglyceride synthesis and how this regulation changes in the obese and diabetic states.